d-Serine Uptake and Release in PC-12 Cells Measured by

Jan 22, 2015 - work, PC-12 cells were incubated with racemic Ser (100 μM ... KEYWORDS: D-Serine, uptake, release, PC-12 cells, chiral analysis, micro...
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Research Article pubs.acs.org/chemneuro

D‑Serine

Uptake and Release in PC-12 Cells Measured by Chiral Microchip Electrophoresis-Mass Spectrometry Xiangtang Li,† Cassandra McCullum,† Shulin Zhao,†,‡ Hankun Hu,†,§ and Yi-Ming Liu*,† †

Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch Street, Jackson, Mississippi 39217, United States College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin 541004, China § Zhongnan Hospital, Wuhan University, Wuhan 430071, China ‡

ABSTRACT: Previous work has established that D-serine (DSer) plays important roles in certain neurological processes. Study on its uptake/storage and release by neuronal cells is highly significant for elucidating relevant mechanisms. In this work, PC-12 cells were incubated with racemic Ser (100 μM each enantiomer). After incubation, both intra- and extracellular levels of D-Ser and L-Ser were quantified by chiral microchip electrophoresis with mass spectrometric detection. It was found the cells preferably took up D-Ser over L-Ser. After 120 min incubation, D-Ser percentage ([D-Ser]/([D-Ser] + [L-Ser]) in the culture media changed from 50% to 9% while inside the cells it increased from 13% to 67%. Small neutral amino acids such as threonine impaired D-Ser uptake. Ser release was studied by using PC-12 cells preloaded with D-Ser. KCl, Glu, and Gly evoked Ser release. Interestingly, while depolarization by KCl evoked release of Ser as a D-Ser/L-Ser mixture of 1:1 ratio, the stereoisomeric composition of Ser released due to Glu exposure varied with the exposure time, ranging from 73% D-Ser (i.e., [D-Ser] > [L-Ser]) at 2 min to 44% (i.e., [D-Ser] < [L-Ser]) at 14 min, clearly indicating a stereochemical preference for D-Ser in Ser release from neuronal cells evoked by Glu-receptor activation. KEYWORDS: D-Serine, uptake, release, PC-12 cells, chiral analysis, microchip electrophoresis-mass spectrometry

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barrier.14,15 Exogenous D-Ser intake may, therefore, also have an influence on its level in the brain. Studies on D-Ser uptake and release in C6 glioma cells, cortical astroglia cells, primary neuronal cultures, and rat brain have been reported. Cultured C6 glioma cells were found to uptake and accumulate both [3H]D- and [3H]L-Ser from culture media in a temperature-dependent and saturable manner. The affinities (km values) were measured to be 2.5 mM for [3H]DSer and 0.11 mM for [3H]L-Ser.16 An alanine-serine-cysteine transporter (ASCT2) mediated mechanism was proposed for DSer uptake in these cells.17 A recent study with cultured cortical astroglia cells prepared from Wistar pups showed that astrocytes contained specific vesicles capable of releasing DSer by Ca2+-dependent exocytosis.13 In contrast with the notion that D-Ser is exclusively released from astrocytes, studies with primary neuronal cultures showed that D-Ser was released by neuronal depolarization both in vitro and in vivo. Veratridine (50 μM) or depolarization by 40 mM KCl elicited a significant release of endogenous D-Ser from the cells while controls with astrocyte cultures showed that glial cells were insensitive to veratridine. Since no internal or external Ca2+ presence required for the release, it was believed that D-Ser was released from these cells by a nonvesicular release mechanism.18,19 Using an

-Serine (D-Ser) is a putative glial neurotransmitter in mammalian central nervous system (CNS).1−4 It binds glutamatergic N-methyl D-aspartate receptors (NMDARs) as an obligatory coagonist, thus modulating their functional activation. In the brain, a deficiency of D-Ser contributes to NMDAR hypofunction involved in neurological conditions such as schizophrenia.5 Therefore, D-Ser replenishment serves as an effective therapeutic strategy.5,6 Many studies have shown that D-Ser in adult rat brain is principally enriched in glial cells, particularly in type-2 astrocytes. Serine racemase (SR) is believed to be the enzyme responsible for D-Ser synthesis from 7,8 L-Ser. SR was initially found in astrocytes and microglia. Further studies showed that SR was actually expressed at a significantly higher level in neurons than in astrocytes.4,9,10 These findings support the theory that D-Ser is synthesized in neurons, and then transported both to other neurons and to surrounding astrocytes where it is stored. However, recent studies comparing SR and D-Ser levels in cell type-specific SR knockout mice found that, despite a significant reduction of SR in neuronal SR knockout mouse brains, D-Ser levels were only minimally reduced, indicating that neurons are not the sole source of D-Ser.11,12 Another study also revealed that SR was present in glial vesicles isolated from astroglial cells.13 Further, it has been shown that intraperitoneal (i.p.) administration of D-Ser can cause a significant increase of D-Ser level in the rat brain, confirming D-Ser is able to pass through the blood-brain © XXXX American Chemical Society

Received: November 30, 2014 Revised: January 17, 2015

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DOI: 10.1021/cn5003122 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 1. Study of D-Ser uptake and release in PC-12 cells with a chiral microchip electrophoresis-mass spectrometric platform: (A) lab-on-chip experimental setup; (B) electropherograms showing L-Ser and D-Ser peaks separated by chiral microchip electrophoresis and detected by selected reaction monitoring (SRM) m/z 88 → 60; and (C) MS2 spectrum confirming the peak identity.

Figure 2. Uptake of Ser by PC-12 neuronal cells: (A) electropherograms obtained from analysis of an incubation solution of PC-12 cells with racemic Ser (at 100 μM each enantiomer) at 20 and 120 min to determine extracellular levels of D-Ser and L-Ser; (B) concentration trends for L-Ser and D-Ser with incubation time; and (C) electropherograms obtained from quantification of intracellular L-Ser and D-Ser enantiomers before (0 min) and after incubation (120 min), respectively.

high-K+ aCSF. NMDA also induced increases in D-Ser, L-Ser, glutamate, and GABA concentrations in the striatum. The increases were attenuated by the NMDA receptor antagonist

online microdialysis-CE-LIF method to measure the D- and LSer levels in the rat striatum, in vivo studies found that extracellular D-Ser level increased in response to application of B

DOI: 10.1021/cn5003122 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience MK-801.20 D-Ser release and uptake were also investigated in vivo using an enzyme-based D-Ser microelectrode implanted in rat brain.21 It was found that small L-amino acids, including LAla and L-Thr, were able not only to block D-Ser reuptake, but also to evoke release of endogenous D-Ser. These findings suggested that small neutral amino acid transporters, including Asc-1 and ASCT-2 might be involved in both release and uptake of D-Ser through an amino acid heteroexchange mechanism. In this study, D-Ser uptake and release in PC-12 neuronal cells were investigated for the first time. Pheochromocytomaderived PC-12 cell line is a well-established in vitro model for studies of regulated secretion in neuronal cells.22 The cell line has been shown to express both monomeric- and dimeric forms of SR, and their biological activity in the cells has been reported.23,24 An advantageous analytical method based on chiral microchip electrophoresis with mass spectrometric detection was used to quantify L- and D-Ser simultaneously. In this method, L-Ser and D-Ser were separated from each other by electrophoresis and then detected by mass spectrometry with specificity. This allowed exploring the stereochemical preference of the pathways for uptake and release of D-Ser in the neuronal cells. Effects of small amino acids, including L-Thr and L-Arg on D-Ser uptake, were studied to verify the involvement of Asc-1 transporter in the uptake. Cellular release of D-Ser under various chemical stimulations was studied using PC-12 cells that were preloaded with D-Ser. Particular attention was given to comparing stereoisomeric compositions of Ser released due to KCl-induced neuronal polarization with that caused by Glu-induced receptor activation in searching for a better understanding of the mechanism for D-Ser release from neuronal cells.

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shortens the analysis time and improves assay accuracy and repeatability. Stereochemical Preference for D-Ser over L-Ser in the Uptake. D-Ser uptake in PC-12 cells was investigated by incubating the cells with racemic Ser (100 μM each enantiomer) in the sample reservoir on the MCE-MS platform (Figure 1A). Racemic Ser instead of D-Ser was used for the incubation because the stereochemical aspects of the uptake pathways could be investigated. At different time intervals, the incubation solution was injected into the MCE-MS analytical platform to simultaneously quantify L-Ser and D-Ser. Typical electropherograms obtained are shown in Figure 2A. The D-Ser peak decreases as the incubation time increases while the L-Ser peak almost remains the same, indicating D-Ser disappears from the incubation medium and L-Ser remains intact during the incubation. The concentration trends measured for the two enantiomers are shown in Figure 2B. There are two possible causes for these results. One is that PC-12 cells selectively metabolize D-Ser while keeping L-Ser intact, and another is they uptake D-Ser preferably over L-Ser. To clarify this point, intracellular levels of D-Ser and L-Ser were determined before and after incubation with racemic Ser. The results (shown in Figure 2C) suggest that the cells uptake D-Ser while leaving LSer alone under the incubation conditions selected. After 120 min incubation with racemic Ser at 100 μM each enantiomer, intracellular level of D -Ser increases substantially. Ser enantiomeric composition inside the cells changed from 13% to 67% D-Ser (calculated by [D-Ser]/([L-Ser] + [D-Ser]). These findings clearly indicate that PC-12 cells uptake D-Ser preferably over L-Ser and accumulate it in the cells for future use. Asc-1 Transporter Involvement and Other Unknown Pathways Involved in D-Ser Uptake. We have shown that PC-12 neuronal cells preferably uptake D-Ser over its antipode, L-Ser from culture media. Small neutral amino acid transporters (i.e., Asc-1 and ASCT2) are known to transport D-Ser in the nervous system. Asc-1 is primarily expressed in neuronal cells and has a high affinity for Ser, Cys, and Thr.21,35−37 To assess Asc-1’s role in D-Ser uptake by PC-12 cells, the cells were incubated with racemic Ser (at 100 μM each enantiomer) in the presence of amino acids at 80 mM. After 120 min incubation, the culture medium was analyzed to determine L-Ser and D-Ser. For each incubation test, the relative concentration of D-Ser (i.e., [D-Ser]/[L-Ser]) was used to assess the effects of the respective amino acid on D-Ser uptake. Four amino acids, including two neutral (Thr and Ala) and two basic (Lys and Arg) were studied. The results are summarized in Figure 3. In the presence of Ala or Thr, very minor decreases in D-Ser concentration are observed as compared with those in the control or Arg- or Lys-containing solutions. Both of the neutral amino acids tested, that is, Ala and Thr, effectively impaired DSer uptake by the cells while the basic amino acids, that is, Arg and Lys, almost showed no impairing effects. These findings firmly indicate that Asc-1 participates in D-Ser uptake by PC-12 neuronal cells, which is in consistence with the previously reported studies. Further, the present study demonstrates that Asc-1 mediated amino acid exchange is not the solo pathway of D-Ser uptake in PC-12 cells. This is because through the amino acid exchange pathway alone, the intracellular D-Ser level should not be higher than that of L-Ser when their extracellular levels are the same or reversed. Nevertheless, as shown above, incubating the cells with racemic Ser (at 100 μM each enantiomer) for 120 min caused a change in intracellular percentage of D-Ser from 13% to 67% D-Ser. These results

RESULTS AND DISCUSSION

To study D-Ser uptake in PC-12 neuronal cells, the cells were incubated with racemic Ser at a concentration of 100 μM each enantiomer. After incubation, intracellular and extracellular levels of both L-Ser and D-Ser were simultaneously quantified by using a chiral microchip electrophoresis-mass spectrometric (MCE-MS) method recently developed in our lab.25,26 To study cellular release of D-Ser, the cells preloaded with D-Ser were incubated with PBS containing various stimulants, and then extracellular levels of both L-Ser and D-Ser were determined. Figure 1A illustrates the MCE-MS analytical platform. Incubation tests were carried out in the sample reservoir on the microfluidic chip. The reservoir was equipped with a piece of 0.22 μm membrane, preventing cells from getting into microfluidic channels. The incubation solution could be injected into the system at selected time intervals for quantifying L-Ser and D-Ser. Under the selected MCE conditions, L-Ser and D-Ser were well separated, and very importantly, the separation was completed within a short time (5 h to detect more L-Ser than D-Ser in the media.

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CONCLUSION PC-12 neuronal cells uptake and accumulate Ser with a stereochemical preference for D-Ser over L-Ser. This is confirmed by quantifying extracellular and intracellular levels of D-Ser and L-Ser after incubating the cells with racemic Ser. This stereochemical preference cannot be explained by Acs-1 mediated amino acid exchange mechanism alone. An unknown pathway is, therefore, speculated for D-Ser transport. Ser release from PC-12 cells is evoked by KCl, Gly, and Glu. While equal amounts of D-Ser and L-Ser are released in both KCl depolarization and Gly-induced amino acid exchange, Gluevoked Ser release through Glu receptor activation exhibits a stereochemical preference for D-Ser.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 601-979-3491. Fax: 601-979-3674. E-mail: yiming.liu@ jsums.edu. Author Contributions

X.L., S.Z., H.H., and Y.-M.L. designed the experiments and analyzed the data. X.L. and C.McC. performed the experiments. X.L. and Y.-M.L. wrote the manuscript. Funding

This study was supported by a grant from U.S. National Institutes of Health (GM089557).

METHODS

Cell Culture and Incubation Tests. PC-12 cells (obtained from ATCC) were cultured in complete RPMI medium supplemented with 10% heat inactivated horse serum and 5% FBS. Cells were routinely subcultured every 4−5 days. To investigate Ser uptake, portions (450 μL) of a cell suspension at 2 × 106 cells/mL were transferred to respective wells of a plate, and to each well 50 μL Ser racemate solution (1 × 10−3 M each enantiomer prepared in PBS at pH 7.4) was added. The plate was kept in an incubator of 5% CO2. At selected time intervals, the incubation solution was stirred by gentle pipetting and a portion (25 μL) was transferred to the sample reservoir in the MCEMS platform for analysis. To study the effects of amino acids on Ser uptake, the respective amino acid was added to Ser racemate solution and the Ser solution was used as described above. To investigate Ser release from PC-12 cells, 450 μL cell suspension (at 2 × 106 cells/mL) was transferred to a plate and then 50 μL Ser racemate solution (1 × 10−3 M each enantiomer prepared in PBS at pH 7.4) was added. The plate was kept in an incubator of 5% CO2 for 90 min. The cells were collected by spinning at 100 rpm, washed twice with PBS buffer, and suspended in 500 mL PBS. To the sample reservoir in the MCE-MS platform 20 μL cell suspension was transferred and 5 μL stimulant solution prepared in PBS was added. The solution was well mixed by

Notes

The authors declare no competing financial interest.

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DOI: 10.1021/cn5003122 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX